AML1-ETO is a DNA binding fusion protein generated from t(8;21)(q22;q22). This chromosomal translocation is one of the most common genetic abnormalities in acute myeloid leukemia (AML), identified in over 10% of all cases. Our own studies and those of others using both human and mouse models have demonstrated that AML1-ETO plays an important role in the development of leukemia. However, it is not sufficient by itself for leukemia development. Interestingly, multiple forms of AML1-ETO are present in t(8;21) leukemia samples due to alternative RNA splicing and deletion mutations. Most of our current understanding of t(8;21) in leukemogenesis comes from studies of the full length AML1-ETO. The role played by these other shorter forms is largely unknown. During the previous funding period, we demonstrated the existence of an alternatively spliced isoform of t(8;21), AML1-ETO9a, which includes one extra exon 9a and generates a fusion protein that lacks 178 amino acids comprising the C-terminus of the full length AML1-ETO. More importantly, AML1-ETO9a is highly leukemogenic in a mouse model of retrovirus vector mediated transduction- hematopoietic cell transplantation. These findings lead to the hypothesis that the most critical domains of AML1-ETO for leukemogenesis are located within the N-terminal portion of this fusion protein covered by AML1-ETO9a, which function via regulation of specific target gene expression and interaction with key complexes involved in basic cellular events. We have developed three specific aims to test this hypothesis. Specific Aim #1 will analyze the effect of AML1-ETO9a on leukemogenesis and hematopoiesis using human hematopoietic cells and using AML1-ETO9a knock-in mice. Specific Aim #2 will study the effect of AML1- ETO9a on leukemogenesis by identifying and characterizing its direct target genes. We have performed combined gene expression-promoter occupancy profiling arrays with primary AML1-ETO9a induced leukemic cells to identify molecular targets modulated by this leukemogenic fusion protein. Specific Aim #3 will characterize proteins interacting with AML1-ETO to understand the molecular mechanism of AML1-ETO involved leukemia development. We have established animal and cell line models and biochemical approaches to pursue these proposed studies. The experiments may provide valuable insight into the molecular mechanisms of leukemogenesis and therapeutic drug designs in cancer treatment. 1